Two signals (Ag and costimulation) are required to activate naive CD8 T cells, but they are not sufficient. Work done during the previous granting period has provided strong evidence that the cells need a third signal in order to undergo clonal expansion and develop effector function and a responsive memory population. Signal three can be provided by IL-12 or Type I IFNs, as demonstrated by in vitro activation, and in vivo by their ability to replace the need for adjuvant in stimulating a response to peptide Ag. Thus, the third signal converts a tolerizing exposure to Ag to effective priming. The aims to be pursued include: Aim 1. To determine if IL-12 and IFN-alpha provide the third signal for naive CD8 T cell activation by dendritic cells and by in vivo challenge with Ag. Experiments will be done to determine if stimulation by activated DC requires IL-12 and/or IFN-alpha, and whether CD40-dependent help from CD4 cells involves stimulation of DC to produce signal 3 cytokines. In vivo experiments will employ cytokine- and receptor-deficient mice to determine the extent to which CTL responses depend upon IL-12 and/or Type I IFNs. Aim 2. To determine the effects of signal 3 cytokines on activation of naive CD8 T cells: gene regulation, survival and effector function. Functional studies and gene array analyses have suggested a number of hypotheses regarding mechanisms by which signal three contributes to activation, and these will be tested. Aim 3. To determine expression in memory cells of genes regulated in naive cells by signal 3 cytokines. Memory cells do not require a third signal to be activated, and the basis for this will be determined. It is anticipated that the results of these studies will contribute novel insights into the requirements for generatingCD8 T cell responses. In addition, the results will have important implications for manipulating these responses for protective and therapeutic immunizations, and potentially for induction of tolerance where it is desirable that responses be prevented.